1. Field of the Invention
The present invention relates generally to novel classes of compounds which interact with adenine nucleotide translocase (ANT), as well as to compositions and methods for using such compounds to treat conditions associated with altered mitochondrial function.
2. Description of the Related Art
Mitochondria are the main energy source in cells of higher organisms, and these organelles provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes. These include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis.
Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix. (For a review, see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s). The cristae, originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of Structural Biology 119:260). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (>˜10 kDa) molecules.
Altered or defective mitochondrial activity, including but not limited to failure at any step of the ETC, may result in the generation of highly reactive free radicals that have the potential of damaging cells and tissues. These free radicals may include reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and potentially other reactive species that may be toxic to cells. For example, oxygen free radical induced lipid peroxidation is a well established pathogenetic mechanism in central nervous system (CNS) injury such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke).
In addition to free radical mediated tissue damage, there are at least two deleterious consequences of exposure to reactive free radicals arising from mitochondrial dysfunction that adversely impact the mitochondria themselves. First, free radical mediated damage may inactivate one or more of the myriad proteins of the ETC. Second, free radical mediated damage may result in catastrophic mitochondrial collapse that has been termed “permeability transition” (PT) or “mitochondrial permeability transition” (MPT). According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential in the inner mitochondrial membrane by a coupled chemiosmotic mechanism, as described herein. Free radical oxidative activity, may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source. In addition, mitochondrial proteins such as cytochrome c may leak out of the mitochondria after MPT and may induce the genetically programmed cell suicide sequence known as apoptosis (Wilson, 1998 Cell Death Differen. 5:646-652) or programmed cell death (PCD).
Altered mitochondrial function characteristic of mitochondria associated diseases may also be related to loss of mitochondrial membrane electrochemical potential by mechanisms other than free radical oxidation, and MPT may result from direct calcium overload due to excitotoxic mechanisms or indirect effects of mitochondrial genes, gene products or related downstream mediator molecules and/or extramitochondrial genes, gene products or related downstream mediators, or from other known or unknown causes. Loss of mitochondrial electrochemical potential therefore may be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases.
Mitochondrial defects, which may include defects related to the discrete mitochondrial genome that resides in mitochondrial DNA and/or to the extramitochondrial genome, which includes nuclear chromosomal DNA and other extramitochondrial DNA, may contribute significantly to the pathogenesis of diseases associated with altered mitochondrial function. For example, alterations in the structural and/or functional properties of mitochondrial components comprised of subunits encoded directly or indirectly by mitochondrial and/or extramitochondrial DNA, including alterations deriving from genetic and/or environmental factors or alterations derived from cellular compensatory mechanisms, may play a role in the pathogenesis of any disease associated with altered mitochondrial function. A number of diseases and conditions are thought to be caused by, or to be associated with, alterations in mitochondrial function. These include: Alzheimer's Disease (AD); diabetes mellitus; obesity; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD); myoclonic epilepsy ragged red fiber syndrome; osteoarthritis; and Friedrich's ataxia. The extensive list of additional diseases associated with altered mitochondrial function continues to expand as aberrant mitochondrial or mitonuclear activities are implicated in particular disease processes.
A hallmark pathology of AD and potentially other diseases associated with altered mitochondrial function is the death of selected cellular populations in particular affected tissues. Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis (also referred to as “programmed cell death” or PCD) in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Fiskum et al., J. Cerebral Blood Flow and Met 19:351-369, 1999; Murphy et al., J. Cerebral Blood Flow and Met. 19:231-245, 1999; Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) and elevated ROS levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42,1994).
Oxidatively stressed mitochondria may release a pre-formed soluble factor that can induce chromosomal condensation, an event preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). In addition, members of the Bcl-2 family of anti-apoptosis gene products are located within the outer mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear to protect membranes from oxidative stress (Korsmeyer et al., Biochim. Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membrane appears to be indispensable for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269: 16521-24, 1994). Thus, change in mitochondrial physiology may be important mediators of apoptosis.
Thus, in addition to their role in energy production in growing cells, mitochondria (or, at least, mitochondrial components) participate in cell death (eg., necrosis and apoptosis) (Newmeyer et al., 1994, Cell 79:353-364; Liu et al., 1996, Cell 86:147-157). Apoptosis is apparently also required for, inter alia, normal development of the nervous system and proper functioning of the immune system. Moreover, some disease states are thought to be associated with either insufficient (e.g., cancer, autoimmune diseases) or excessive (e.g., stroke damage, AD-associated neurodegeneration) levels of apoptosis. For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed (1998, Science 281:1309-1312), Green (1998, Cell 94:695-698) and Kromer (1997, Nature Medicine 3:614-620). Hence, agents that effect apoptotic events, including those associated with mitochondrial components, might have a variety of palliative, prophylactic and therapeutic uses.
The adenine nucleotide translocase (ANT), a nuclear encoded mitochondrial protein, is reportedly the most abundant protein of the inner mitochondrial membrane, forming dimers that comprise up to 10% of the total mitochondrial protein in highly oxidative tissue like cardiac and skeletal muscle. Three human ANT isoforms have been described that appear to differ in their tissue expression patterns, and other mammalian ANT homologues have been described. See, e.g., Wallace et al., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 283-307, and references cited therein. ANT proteins mediate the exchange across the mitochondrial inner membrane of ATP synthesized in the mitochondrial matrix for adenosine diphosphate (ADP) in the cytosol. This nucleotide exchange is the most active transport system in aerobic cells, and is a critical component in maintaining cellular energy metabolism (for a review see Klingenberg, J. Bioenergetics and Biomembranes 25:447-457, 1993). ANT has also been implicated as an important molecular component of the MPT pore, a Ca2+-regulated inner membrane channel that plays an important modulating role in apoptotic processes.
By way of background, all five of the multisubunit complexes that mediate ETC activity are localized to the inner mitochondrial membrane. ANT represents the most abundant of the inner mitochondrial membrane proteins. In at least three distinct chemical reactions known to take place within the ETC, positively-charged protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibria of charged species creates an electrochemical potential of approximately 220 mV referred to as the “protonmotive force” (PMF), which is often represented by the notation Δψ and represents the sum of the electric potential and the pH differential across the inner mitochondrial membrane (see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s and references cited therein).
This membrane potential drives the ANT-mediated stoichiometric exchange of ATP and ADP across the inner mitochondrial membrane, and provides the energy contributed to the phosphate bond created when ADP is phosphorylated to yield ATP by ETC Complex V, a process that is “coupled” stoichiometrically with transport of a proton into the matrix. Under normal metabolic conditions, the inner membrane is impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the sole means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during MPT, which may accompany a disease associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby “uncoupling” respiration because electron transfer and associated proton pumping yields no ATP. Thus, MPT involves the opening of a mitochondrial membrane “pore”, a process by which, inter alia, the ETC and ATP synthesis are uncoupled, Δψm collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca2+, Na+, K+, H+) and large (e.g., proteins) molecules.
Without wishing to be bound by theory, it is unresolved whether this pore is a physically discrete conduit that is formed in mitochondrial membranes, for example by assembly or aggregation of particular mitochondrial and/or cytosolic proteins and possibly other molecular species, or whether the opening of the “pore” may simply represent a general increase in the porosity of the mitochondrial membrane.
MPT may also be induced or blocked by compounds that bind one or more mitochondrial molecular components. Such compounds include, but are not limited to, atractyloside and bongkrekic acid, which are known to bind to ANT. Methods of determining appropriate amounts of such compounds to induce MPT are known in the art (see, e.g., Beutner et al., 1998 Biochim. Biophys. Acta 1368:7; Obatomi and Bach, 1996 Toxicol. Lett. 89:155; Green and Reed, 1998 Science 281:1309; Kroemer et al., 1998 Annu. Rev. Physiol. 60:619; and references cited therein). Thus, certain mitochondrial molecular components, such as ANT, may contribute to the MPT mechanism.
It is known that when fatty acids bind to ANT, they can induce what is termed “mild” mitochondrial uncoupling. In bioenergetic terms, the word “mild” means that the uncoupling is only evident at the resting state of the mitochondria (i.e. state 4, nonphosphorylating respiration) when the membrane potential is maximal, and that there is little or no effect during robust ATP production. Additionally, it has been discovered that this uncoupling induced by free fatty acids may be reversed by the addition of the ANT ligand carboxyatractyloside (see e.g., Andreyev et al., 1989 Eur. J. Biochem. 182:585-592; Skulachev, 1991 FEBS Lett. 294:158-162; Skulachev, 1996 FEBS Lett. 397:7-10; Korshunov et al., 1998 FEBS Lett. 435:215-218; Wojtczak et al., 1998 Archives of Biochem. and BioPhys. 357:76-84; and references cited therein), suggesting that carboxyatractyloside blocks the proton conductance induced by the free fatty acid. Since the high membrane potential in the resting state of the mitochondria potentiates mitochondrial free radical production (see e.g., Boveris and Chance, 1973 Biochem. J. 134:707-716; Korshunov et al., 1997 FEBS Lett. 416:15-18; and references cited therein), it has been theorized that periods of mild uncoupling may serve the purpose of reducing oxidative stress and could slow the rate of Ca2+ uptake at high membrane potential (Skulachev, 1996 FEBS Lett. 397:7-10; Korshunov et al., 1997 FEBS Lett. 416:15-18; and references cited therein).
ANT proteins, as well as other transporter proteins known more generally as uncoupling proteins (UCPs), belong to a larger family of proteins known as the “carrier” family. The theory that mild uncoupling may be induced via brain-specific isoforms of UCPs has recently become the focus of several studies (Yu et al., 2000 FASEB J. 14(11):1611-1618; Farrelly et al., 2001 Analytical Biochem. 293:269-276; and references cited therein). Additionally, mild mitochondrial uncoupling has recently been proposed as a possible treatment for ischemia-reperfusion injury (Morin et al., 2001 Advanced Drug Delivery Rev. 49:151-174; and references cited therein).
Clearly there is a need for compounds and methods that limit or prevent damage to organelles, cells and tissues that may directly or indirectly result from alterations in mitochondrial function including mitochondrial dysfunction, such as mitochondrial permeability transition that is the cause or consequence of oxidative phosphorylation uncoupling and/or intracellular free radical generation. Accordingly, while significant advances have been made in this field, there is still a need in the art for small molecules that will bind, form a complex with, or otherwise interact with ANT. There is also a need for pharmaceutical compositions containing the same, as well as methods relating to the use thereof to treat conditions associated with altered mitochondrial function. The present invention fulfills these needs and provides other related advantages.